Pcr primer pair for identification of ommastrephes bartramii in the north pacific ocean based on environmental dna, and method for identifying ommastrephes bartramii using the same

Abstract

A primer pair for identification of Ommastrephes bartramii in the North Pacific Ocean based on environmental DNA, and a method for identifying Ommastrephes bartramii using the same. The primer pair consists of a forward primer OMBA-F: 5′-CGAAGGTTAATCTGTCTCCATCT-3′ (SEQ ID NO: 1), and a reverse primer OMBA-R: 5′-CCCAATTAAAATTTATATACCACCACCT-3′ (SEQ ID NO: 2). A DNA molecular marker locus for identifying Ommastrephes bartramii is located at 16S rRNA of Ommastrephes bartramii. The environmental DNA is amplified and sequenced, and if the obtained sequence is completely identical to the sequence of the DNA molecular marker locus, it is indicated that there is Ommastrephes bartramii in the sampling region.

What is claimed is: 
     
         1 . A PCR primer pair for identification of  Ommastrephes bartramii  in the North Pacific Ocean based on environmental DNA, comprising: 
       
         
           
                 
                 
               
                     
                   a forward primer OMBA-F: 
                 
                     
                   (SEQ ID NO: 1) 
                 
                     
                   5′-CGAAGGTTAATCTGTCTCCATCT-3′; 
                 
                     
                   and 
                 
                     
                     
                 
                     
                   a reverse primer OMBA-R: 
                 
                     
                   (SEQ ID NO: 2) 
                 
                     
                   5′-CCCAATTAAAATTTATATACCACCACCT-3′. 
                 
             
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         2 . A method for identifying  Ommastrephes bartramii  in a region of the North Pacific based on environmental DNA using the primer pair of  claim 1 , comprising:
 (1) subjecting the environmental DNA to PCR amplification using the primer pair to obtain an amplification product; and   (2) comparing the amplification product with a sequence of a DNA molecular marker locus to determine whether there is  Ommastrephes bartramii  in the region;   wherein if the amplification product is identical to the sequence of the DNA molecular marker locus, it is indicated that there is  Ommastrephes bartramii  in the region of the North Pacific Ocean;   the DNA molecular marker locus is located on 16S rRNA of the  Ommastrephes bartramii ; and   the sequence of the DNA molecular marker locus is shown in SEQ ID NO: 3 with a length of 155 bp.   
     
     
         3 . The method of  claim 2 , wherein the environmental DNA is obtained through steps of:
 (a) collecting a water sample from the region of the North Pacific Ocean;   filtering the water sample with a nitrocellulose filter with a diameter of 47 mm and a pore size of 0.4 μm; and storing the nitrocellulose filter at −20° C. for use; and   (b) extracting the environmental DNA from the nitrocellulose filter with a DNeasy Blood & Tissue kit (Qiagen); and storing the environmental DNA at −20° C. for use.   
     
     
         4 . The method of  claim 2 , wherein the step (2) comprises:
 subjecting the amplification product to electrophoresis with 2% agarose gel to obtain a positive band;   subjecting the positive band to DNA sequencing to obtain a sequence of the environmental DNA; and   comparing the sequence of the environmental DNA with the sequence of the DNA molecular marker locus to determine whether there is  Ommastrephes bartramii  in the region.   
     
     
         5 . The method of  claim 2 , wherein a system of the PCR amplification has a volume of 20 μL and consists of:
 2 μL of the environmental DNA as template; 
 15 μL of a PCR Mix; 
 1 μL of 10 μM forward primer OMBA-F; 
 1 μL of 10 μM reverse primer OMBA-R; and 
 ultrapure water. 
 
     
     
         6 . The method of  claim 2 , wherein the PCR amplification is programmed as follows:
 50° C. for 2 min;   95° C. for 10 min; and   55 cycles, each consisting of 95° C. for 15 s and 60° C. for 1 min.